Raniero Rocchi

Glycodermorphins: opioid peptides with potent and prolonged analgesic activity and enhanced blood-brain barrier penetration

1 In order to improve the in vivo stability of the opioid peptide dermorphin we synthesized O‐βglucosylated analogs ([Ser7‐O‐βGlc]dermorphin and [Ser7‐O‐βGlc(Ac)4]‐dermorphin) and C‐αgalactosylated analogs ([Ala7‐C‐αGal]dermorphin and [Ala7‐C‐αGal(Ac)4]‐dermorphin). 2 O‐ and C‐glycosylation of dermorphin halved the peptide affinity for brain μ‐opioid receptors and the biological potency in guinea‐pig ileum assay (GPI). Despite their lower opioid receptor affinity, when administered intracerebroventricularly (i.c.v., 8–40 pmol) and subcutaneously (s.c., 0.5–3 μmol kg−1) in rats, glycosylated analogs were two times more potent than dermorphin in reducing the nociceptive response to radiant heat. Acetylation of sugar hydroxyl groups reduces 5–10 times both biological activity on GPI and μ‐receptor affinity, whereas the antinociceptive potency was equal to (i.c.v.) or only two‐three times lower (s.c.) than dermorphin potency. 3 Blood‐Brain Barrier Permeability Index (BBB‐PI) of the glycodermorphins was significantly higher than that of dermorphin, indicating a facilitated entry into the brain: O‐β‐linked glucoconiugates are expected to enter CNS by the glucose transporter GLUT‐1 of the endothelial barrier. However the calculated BBB‐PI for the C‐αgalactoside was about two times higher than that of the O‐βglucoside, excluding the implication of GLUT‐1 that is known to be selective for O‐β‐links and preferring for the exose glucose. 4 The enhanced brain permeability with the subsequent decrease in peripheral dosage of these opioid peptides did not result in lowering constipation.

Solid phase synthesis and conformation of sequential glycosylated polytripeptide sequences related to antifreeze glycoproteins

Sequential glycopeptides [Thr(beta-D-galactose)-Ala-Ala]n, with n ranging from 2 to 7, as models of natural antifreeze glycoproteins were synthesized by the continuous flow, solid phase procedure. The conformational properties of these materials in solution were investigated by c.d. and 1H-n.m.r. spectroscopy. In aqueous solution the c.d. pattern is practically independent of chain length and is very similar to that of natural antifreeze glycoproteins. The results are interpreted in terms of random coil structure. The absence of ordered structures is further confirmed by n.m.r. data. A small amount of ordered conformation can be induced either by increasing the temperature of the aqueous solution or by addition of TFE. The c.d. pattern of all glycopeptides in water at temperatures higher than 50 degrees C are compatible with the presence of a small amount of alpha-helix or 3(10) helix. Since the glyco-hexapeptide is too short to form an alpha-helix, the hypothesis is made that in the glycopeptides in water at high temperature a small amount of 3(10) helix is formed. The same is observed for the 21-residue glycopeptide in presence of 85% (v/v) TFE. In this medium, the c.d. data on the glyco-hexapeptide are more compatible with the presence of a small amount of beta-structure.

Substitution of the arginine/leucine residues in apidaecin Ib with peptoid residues: effect on antimicrobial activity, cellular uptake, and proteolytic degradation.

Several aspects of the mechanism of action of Pro-rich antimicrobial peptides, together with their low toxicity in mammalian cells, make them good candidates for the development of new antibiotic agents. We investigated the effect induced in the insect antimicrobial peptide apidaecin Ib by the replacement of a single arginine/leucine residue with a N-substituted glycine. The resulting peptoid-peptide hybrids are more resistant to proteolysis and devoid of any significant cytotoxic activity, but moving the [NArg]residue from the N- to the C-terminal end of the molecule progressively reduces the antibacterial activity. Cell uptake experiments in E. coli cells suggest that the loss of antibacterial activity of [NArg(17)]apidaecin is a consequence of its inability to translocate into bacterial cells. Conversely, apidaecin and its peptoid-peptide hybrids are able to cross the plasma membrane in eukaryotic cells and to diffuse in the cytosol, although their translocating ability is far less effective than that of other known cell permeant peptides.

Opioid peptides: synthesis and biological properties of [(Nγ-glucosyl,Nγ-methoxy)-α,γ-diamino-(S)-butanoyl]4-deltorphin-1-neoglycopeptide and related analogues

The [D-Ala2]deltorphin 1 sequence in which the aspartic acid residue is replaced by the N gamma-OCH3-alpha, gamma-diamino (S) butanoyl residue was synthesized using the Fmoc-chemistry-based solid phase procedure. The resulting deltorphin analogue was chemoselectively glucosylated by reaction with unprotected D-glucose (Glc). The Asn4-, (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-galactopyranosyl)-Asn4- and the (2-acetamido-2-deoxy-D-galactopyranosyl)-Asn4-deltorphin I were also prepared for comparison. The affinity of the new compounds for the delta-opioid receptor was expressed by the inhibition constant (Ki) of the binding of the delta-receptor selective ligand [3H]naltrindole (NTI) to rat brain membrane preparations. The in vitro biological activity of the synthetic peptides was compared with that of the mu-opioid receptor agonist dermorphin in guinea pig ileum (GPI) preparations and with that of the delta-opioid receptor agonist deltorphin I in mouse vas deferens (MVD) preparations. The substitution of Asp4 with Asn failed to affect drastically the Ki and IC50 values for delta-sites, suggesting that an electrostatic interaction does not play an essential role in the binding to delta-opioid sites. The steric hindrance of the side chain of the residue in position 4 affects binding to delta-sites. The increase of the Ki value is smaller when the sugar-peptide linkage involves the gamma-nitrogen of the Dab residue in comparison with the Asn amide side chain.

Threonine6‐bradykinin: Conformational study of a flexible peptide in dimethyl sulfoxide by NMR and ensemble calculations

The conformation of the natural peptide threonine6 (Thr6)‐bradykinin, Arg1‐Pro2‐Pro3‐Gly4‐Phe5‐Thr6‐Pro7‐Phe8‐Arg9, was investigated in DMSO by nmr spectroscopy and computer simulations. The structural analysis of the Thr6‐peptide is made particularly interesting by the fact that despite the high sequence homology with native bradykinin (only one conservative substitution: Ser6/Thr6) there is a marked and significant difference in the biological profiles of the two peptides.

N-alkylation of amino acids during hydrogenolytic deprotection

Abstract Partial N-alkylation of amino acids was observed during hydrogenolytic deprotection.

Helix induction potential of N-terminal α-methyl, α-amino acids

A series of longer analogues of the C-peptide of RNAse A has been synthesized with the aim of assessing the helix induction potential in water of α-methyl, α-amino acids at the N-terminus of the chain. The circular dichroism data indicate that one isovaline residue is effective in increasing the helix content of the 13-residue peptide by about 7%.

Comparison of the immunogenicity of wasp venom peptides with or without carbohydrate moieties

Three synthetic vespulakinin analogues either with or without carbohydrate moieties and mastoparan B isolated from Vespa basalis venom were investigated for their immunogenic activity and solution conformation. Mice immunized with these wasp venom peptides, with the exception of (Gal alpha)Thr3, (Gal alpha)Thr4-vespulakinin 1, showed positive antibody responses. However, the response elicited by mastoparan B was much higher than those induced by vespulakinin analogues. The class of antibody induced by these peptides was identified as an IgG1 isotype with kappa-light chain, suggesting stimulation of a T-cell-dependent immune response by these peptides. According to the circular dichroism spectra of these peptides, the structures of the vespulakinin analogues in solution were largely unordered, while mastoparan B exhibited a conformation rich in alpha-helices. The presence of carbohydrate moieties and the rather random structure in vespulakinins may interfere with T-cell recognition of the peptides, leading to lower immune responses.

Influence of Glycosylation on the Conformational Preferences of Folded Oligopeptides

Abstract Synthesis, characterization, and conformational analysis by FT-IR absorption, 1H NMR and X-ray diffraction techniques are described for a series of side-chain O-glycosylated Thr peptides of different main-chain length rich in the helicogenic Aib residue. The results obtained, compared with those of related peptides containing side-chain protected Thr and Ser residues and host Aib homo-oligomers, also reported in this work, provided new information on the preferred conformation of the naturally occurring antifreeze glycopeptides.

Coordination and reactivity of benzyloxycarbonyl-Ala(CN)OR (R = H, CH3) in complexes of platinum(II)

Abstract The conversion of a nitrile to an oxazoline group in an amino acid side chain promoted by platinum(II) was investigated. While benzyloxycarbonyl-β-cyano-alanine (Z-Ala(CN)OH) did not give any evidence of coordination of the nitrile group to Pt(II) in different complexes, the corresponding methyl ester Z-Ala(CN)OCH 3 readily afforded the Pt(II)-nitrile complex trans -[Pt(CF 3 ){Z-Ala(CN)OCH 3 }(PPh 3 ) 2 ][BF 4 ] in good yield by reaction with the cationic derivative trans -[Pt(CF 3 ) 2 (solv)][BF 4 ] (solv = CH 2 Cl 2 ). The reactivity of the CN group in the complex trans -[Pt(CF 3 ){Z-Ala(CN)OCH 3 }(PPh 3 ) 2 ][BF 4 ] with ClCH 2 CH 2 O − to the oxazoline ligand 2 was examined. The presence of the oxazoline ring in the final products was evidenced by IR, 1 H and 13 C NMR data and confirmed by FAB and MALDI mass spectra, but its isolation was hampered by the cleavage of the oxazoline ring under the chromatographic conditions applied.